Method of Treating or Preventing Pathologic Effects of Acute Increases in Hyperglycemia and/or Acute Increases of Free Fatty Acid Flux

ABSTRACT

One aspect of the present invention relates to a method of treating or preventing pathologic effects of hyperglycemia and/or increased fatty acid flux in a subject in need of such treatment or preventive therapy. This method involves administering a composition containing a therapeutically effective amount of a ROS inhibitor to a subject in need thereof.

This application is a continuation in part of U.S. Ser. No. 11/136,254filed May 24, 2005, which claims benefit to and priority from U.S.Provisional Patent Application Ser. No. 60/573,947, filed May 24, 2004.

FIELD OF THE INVENTION

The present invention relates to a method of treating or preventingpathologic effects of acute increases in hyperglycemia and/or acuteincreases of fatty acid flux in a subject.

BACKGROUND OF THE INVENTION

Cardiovascular Complications Associated with Diabetes are a Major PublicHealth Problem.

Diabetes mellitus is an epidemic in the United States (Brownlee,“Biochemistry and Molecular Cell Biology of Diabetic Complications,”Nature 414:813-20 (2001); Nishikawa et al., “Normalizing MitochondrialSuperoxide Production Blocks Three Pathways of Hyperglycaemic Damage,”Nature 404:787-90 (2000); Zimmet et al., “Global and SocietalImplications of the Diabetes Epidemic,” Nature 414:782-7 (2001)).Currently 15-17 million adults (5% of the adult population) in the U.S.are affected by Type I and Type II diabetes (Harris et al., “Prevalenceof Diabetes, Impaired Fasting Glucose, and Impaired Glucose Tolerance inU.S. Adults. The Third National Health and Nutrition Examination Survey,1918-1994,” Diabetes Care 21:518-24 (1998); AD Association, “EconomicCosts of Diabetes in the U.S. in 2002,” Diabetes Care 26:917-932(2003)). By the year 2020, the diabetic population is expected toincrease by another 44% (AD Association, “Economic Costs of Diabetes inthe U.S. in 2002,” Diabetes Care 26.917-932 (2003)). In addition tothose with diabetes mellitus, an additional number of people display themetabolic syndrome, with impaired glucose and insulin tolerance andaltered vascular reactivity.

The greatest impact of diabetes is on the vascular system (Caro et al.,“Lifetime Costs of Complications Resulting From Type 2 Diabetes in theU.S. Diabetes Care 25:476-81 (2002)). Diabetic patients have anincreased risk for vascular disease affecting the heart, brain, andperipheral vessels (Howard et al., “Prevention Conference VI: Diabetesand Cardiovascular Disease: Writing Group I: Epidemiology,” Circulation105:e132-7 (2002)). The relative risk of cardiovascular disease indiabetics is 2-8 times higher than age-matched controls (Howard at al.,“Prevention Conference VI: Diabetes and Cardiovascular Disease: WritingGroup I: Epidemiology,” Circulation 105:e132-7 (2002)). Diabetesaccounts for 180 billion dollars in annual health costs in the U.S.,with 85% of this amount attributable to vascular complications (Caro atal., “Lifetime Costs of Complications Resulting From Type 2 Diabetes inthe U.S. Diabetes Care 25:476-81 (2002)). Indeed, if macrovascularcomplications (stroke, MI, TIA, angina) and microvascular complications(nephropathy, neuropathy, retinopathy, wound healing) are consideredtogether, the vast majority of diabetes related healthcare expendituresresult from vasculopathies.

One or the Reasons why Diabetic Patients have Poor Outcomes is Becauseof Impaired Compensatory Vascular Growth.

The recognition that diabetes impairs survival after ischemic eventsdates back to the last century and has been independently confirmed bytwo landmark epidemiologic studies (The Framingham Study and TheDiabetes Control and Complications Trial) (Garcia et al, “Morbidity andMortality in Diabetics in the Framingham Population. Sixteen YearFollow-Up Study,” Diabetes 23:105-11 (1974); TDCaCTR Group, “The Effectof Intensive Treatment of Diabetes on the Development and Progression ofLong-Term Complications in Insulin-Dependent Diabetes Mellitus,” N EnglJ Med 329:977-86 (1993)). These prospective studies substantiated arelationship between poor glycemic control and decreased survival aftermyocardial infarction. Of note, these trials demonstrated that inaddition to an increased incidence of ischemic episodes (Kannel at al.,“Diabetes and Cardiovascular Risk Factors: the Framingham Study,”Circulation; 59:8-13 (1979)), diabetic patients have higher rates ofpost-infarct complications, such as cardiac failure and secondaryischemic events (Haffner et al., “Mortality From Coronary Heart Diseasein Subjects With Type 2 Diabetes and in Nondiabetic Subjects With andWithout Prior Myocardial Infarction,” N Engl J Med 339:229-34 (1998);Zuanetti et al., “Influence of Diabetes on Mortality in Acute MyocardialInfarction: Data From the GISSI-2 Study,” J Am Coll Cardiol 22:1788-94(1993)). These disparities were not due to increased infarct size in thediabetic population (Wilson, “Diabetes Mellitus and Coronary HeartDisease,” Am J Kidney Dis 32:S89-100 (1998)), suggesting that animpairment existed in the compensatory response of the diabeticmyocardium. Similar impairments have been described in other diabetictissues, including the extremities and brain (Uusitupa at al., “5-YearIncidence of Atherosclerotic Vascular Disease in Relation to GeneralRisk Factors, Insulin Level, and Abnormalities in LipoproteinComposition in Non-Insulin-Dependent Diabetic and Nondiabetic Subjects,”Circulation 82:27-36 (1990); Jude et al., “Peripheral Arterial Diseasein Diabetic and Nondiabetic Patients: a Comparison of Severity andOutcome,” Diabetes Care 24:1433-7 (2001); Tuomilehto et al., “DiabetesMellitus as a Risk Factor for Death From Stroke. Prospective Study ofthe Middle-Aged Finnish Population,” Stroke 27:210-5 (1996)).

The concept that these impairments result from a poorly adaptingdiabetic vasculature has both clinical and experimental support. Sinceangiogenesis and collateral development are the processes that restoreblood flow to watershed areas of the heart, the rapid restoration of anormal vascular density in the microvasculature ultimately determinespatient outcome following ischemia (Helfant et al., “FunctionalImportance of the Human Coronary Collateral Circulation,” N Engl J Med284:1277-81 (1971); Chilian at al., “Microvascular Occlusions PromoteCoronary Collateral Growth,” Am J Physiol 258:H1103-1 (1990)). Indeed,the theoretical basis for therapeutic angiogenesis is the belief thataugmenting the microvascular network in ischemic and watershed areas ofthe heart would be beneficial. Clinical as well as experimental studiesprovide conclusive evidence that diabetes impairs ischemia-drivenneovascularization (Abaci at al., “Effect of Diabetes Mellitus onFormation of Coronary Collateral Vessels,” Circulation 99.2239-42(1999); Tooke, “Microvasculature in Diabetes,” Cardiovasc Res 32:764-71(1996); Waltenberger, “Impaired Collateral Vessel Development inDiabetes: Potential Cellular Mechanisms and Therapeutic Implications,”Cardiovasc Res 49554-60 (2001); Yarom et al., “Human CoronaryMicrovessels in Diabetes and Ischaemia. Morphometric Study of AutopsyMaterial,” J Pathol 166:265-70 (1992)). In animal studies, diabeticanimals demonstrate a decreased vascular density following hindlimbischemia (Rivard et al., “Rescue of Diabetes-Related Impairment ofAngiogenesis By Intramuscular Gene Therapy With Adeno-VEGF,” Am J Pathol154:355-63 (1999); Taniyama et al., “Therapeutic Angiogenesis Induced ByHuman Hepatocyte Growth Factor Gene in Rat Diabetic Hind Limb IschemiaModel: Molecular Mechanisms of Delayed Angiogenesis in-Diabetes,”Circulation 104:2344-50 (2001); Schatteman et al., “Blood-DerivedAngioblasts Accelerate Blood-Flow Restoration in Diabetic Mice” J ClinInvest 106:571-8 (2000)). Human angiographic studies have demonstratedthat diabetic patients have fewer collateral vessels than non-diabeticcontrols (Abaci et al., “Effect of Diabetes Mellitus on Formation ofCoronary Collateral Vessels,” Circulation 99:2239-42 (1999)). Moreover,revascularization via coronary angioplasty, coronary artery bypasssurgery, or lower extremity revascularization has a significantly lowersuccess rate in diabetic patients even in the presence of a patientbypass conduit, suggesting the existence of a defect at themicrocirculatory level (Kip et al., “Coronary Angioplasty in DiabeticPatients. The National Heart, Lung, and Blood Institute PercutaneousTransluminal Coronary Angioplasty Registry,” Circulation 94:1.818-25(1996); Palumbo et al., “Diabetes Mellitus: Incidence, Prevalence,Survivorship, and Causes of Death in Rochester, Minn., 1945-1970,”Diabetes 25:366-73 (1976); Schwartz et al., “Coronary Bypass GraftPatency in Patients With Diabetes in the Bypass AngioplastyRevascularization Investigation (BART),” Circulation 10:2652-8 (2002);Kip et al., “Differential Influence of Diabetes Mellitus on IncreasedJeopardized Myocardium After Initial Angioplasty or Bypass Surgery:Bypass Angioplasty Revascularization Investigation,” Circulation105:1914-20 (2002)).

TABLE 1 Published Studies Supporting Impaired Ischemic Responsiveness inDiabetes Type of Study Study Major Findings Abaci et al^((a)) ClinicalAngiographic demonstration of decreased collaterals in the hearts ofdiabetic patients Abaci et al^((b)) Clinical Cardiac failure is moreCommon following an M1 in diabetic patients Altavilla et ExperimentalDiabetic mice have less VEGF, less angiogenesis and impaired woundal^((c)) healing compared to normal mice Arora et al^((d)) ClinicalDiabetics undergoing lower-extremity bypass maintain an impairedvascular reactivity even after successful surgical grafting,highlighting the limits of surgical interventions Bradley et ClinicalDiabetic patients have worse survival after an M1 al^((e)) Chou etal^((f)) Experimental First demonstration that myocardial tissue andcells from diabetic animals express less VEGF and its receptors Frank etal^((g)) Experimental Diabetic mice express much less VEGF RNA andprotein in their wounds Goova et al^((h)) Experimental Blockade of theRAGE receptor accelerated wound healing, augmented VEGF expression, andincreased angiogenesis in diabetic mice. Guzik et al^((i)) ClinicalBlood vessels from diabetic patients produce augmented levels ofsuperoxide, a marker/cause of oxidative stress Haffner et ClinicalDiabetic patients have a greatly increased incidence of experiencing anM1 al^((j)) and dying from an M1 Hiller et al^((k)) ClinicalEpidemiologic study suggesting that diabetic microangiopathy is greatlyincreased in diabetics Jude et al^((l)) Clinical Diabetic patients havean increased incidence, severity, and poorer outcomes in peripheralarterial disease of the lower extremities Kip et al^((m)) ClinicalAngiographic and epidemiologic study demonstrating that diabeticpatients have more diffuse atherosclerotic disease, and worm outcomesafter seemingly successful interventional revascularization Lerman etExperimental First demonstration that cells isolated from diabeticanimals and patients al^((n)) produce attenuated levels of VEGF inhypoxia Marsh et al^((o)) Experimental Monocytes from diabetic patientswithout retinopathy express less VEGF in hypoxia compared to monocytesfrom patients with diabetic retinopathy Partamian et Clinical Diabeticpatients have increased peri-infarct complications and decreasedal^((p)) long-term survival Rivard et al^((q)) Experimental Diabetesdecreases reactive angiogenesis and tissue survival following hindlimbischemia Schatteman Experimental Angioblasts from diabetic humans showdecreased proliferation and et al^((r)) differentiation to matureendothelial cells in culture. Also, diabetic mice have less tolerancehindlimb ischemia than nondiabetic mice Tepper et al^((s)) ExperimentalFirst demonstration that endothelial progenitor cells from diabeticpatients show decreased function with assays that measure functionsimportant for angiogenesis Yarom et al^((t)) Clinical Autopsy pathologicstudy demonstrating that diabetic patients have decreasedischemia-induced reactive angiogenesis ^((a))Abaci et al., “Effect ofDiabetes Mellitus on Formation of coronary Collateral Vessels,”Circulation 99: 2239-42 (1999). ^((b))Abbort et al., The Impact ofDiabetes on Survival Following Myocardial Infarction in Men vs Women.Framiogham Study,” Jama 260: 3456-60 (1988). ^((c))Altavilla et at.,“Inhibition of Lipid Peroxidation Restores Impaired Vascular EndothelialGrowth Factor Expression and Stimulates Wound Healing and Angiogenesisin the Genetically Disbetic Mouse,” Diabetes 50: 667-74 (2001).^((d))Arora et al., “Cutaneous Microcirculation in the NeuropathicDiabetic Foot Improves: Significantly But Not Completely AfterSuccessful Lower Extremity Revascularization,” J Yasc Surg 35: 501-5(2002) ^((e)) Bradley et al., “Survival of Diabetic Patients AfterMyocardial Infarction,” Am J Med 20: 207-216 (1956). ^((f))Chou et al.,“Decreased Cardiac Expression of Vascular Endothelial Growth Factor andits Receptors in Insulin-Resistant and Diabetic States. A PossibleExplanation for Impaired Collateral Formation in Cardiac Tissue.”Circulation 105: 373-9 (2002). ^((g))Frank et al., “Regulation ofVascular Endothelial Growth Factor Expression in Cultured KeratinocytesImplications for Normal and Impaired Wound Healing,” J Biol Chem 270:12607-13 (1995). ^((h))Goova et at., “Blockade of Receptor for AdvancedGlycation End-Products Restores Effective Wound Healing in DiabeticMice,” Am J Pathol 159: 513-25 (2001). ^((i))Guzik et al., “Mechanismsof Increased Vascular Superoxide Production in Human Diabetes Mellitus.Role of NAD(P)H Oxidase and Endothelial Nitric Oxide Synthase,”Circulation 105: 1656-62 (2002) ^((j))Haffner et al., “Mortality FromCoronary Heart Disease in Subject With Type 2 Diabetes and inNondiabetic Subjects With and Without Prior Myocardial Infarction,” NEngl J Med 339: 229-34 (1998). ^((k))Hiller et al., “DiabeticRetinopathy and Cardiovascular Disease in Type II Diabetics. TheFramingham Heart Study and the Framingham Eye Study,” Am J Epidemiol128: 402-9 (1988). ^((l))Jude et al., “Peripheral Arterial Disease inDiabetic and Nondiabetic Patients; a Comparison of Severity andOutcome,” Diabetes Care 24: 1433-7 (2001). ^((m))Kip et al., “CoronaryAngioplasty in Diabetic Patients. The National Heart, Lung, and BloodInstitute Percutaneous Transluminal Coronary Angioplasty Registry,”Circulation 94: 1818-25 (1996) ^((n))Lerman et al., “CellularDysfunction in the Diabetic Fibroblast; Impairment in Migration,Vascular Endtothelial Growth Factor Production, and Response toHypoxin,” Am JPathol 162: 303-12 (2003). ^((o))Marsh et al., “HypoxicInduction or Vascular Endothelial Growth Factor is Markedly Decreased inDiabetic Individuals Who Do Not Develop Retinopathy,” Diabetes Care 23:1375-80 (2000). ^((p))Partamian et al., “Acute Myocardiol Infarction in258 Cases of Diabetes, Immediate Mortality and Five-Year Survival.” NEngl J Med 273: 455-61 (1965). ^((q))Rivard et al., “Rescue ofDiabetes-Related Impairment of Angiogenesis By Intramuscular GeneTherapy With Adeno-VEGF,” Am J Pathol 154: 355-63 (1999)^((r))Schatteman et al., “Blood-Derived Angioblasts AccelerateBlood-Flow Restoration in Diabetic Mice,” J Clin Invest 106: 571-8(2000). ^((s))Tepper et al., “Human Endothelial Progenitor Cells FromType II Diabetics Exhibit Impaired Proliferation, Adhesion, andIncorporation Into Vascular Structures,” Circulation 106: 2781-6 (2002).^((t))Despite the preponderance of these observations, the mechanismsunderlying impaired neovascularization in diabetes remain unclear.Impaired VEGF expression has been implicated as a significantcontributing factor (Rivard et al., “Rescue of Diabetes-RelatedImpairment of Angiogenesis By Intramuscular Gene Therapy WithAdeno-VEGF,” Am J Pathol 154.355-63 (1999); Schratzberger, et al.,“Reversal of Experimental Diabetic Neuropathy by VEGF Gene Transfer,” JClin Invest 107:108392 (2001); Aiello et al., “Role of VascularEndothelial Growth Factor in Diabetic Vascular Complications,” KidneyInt Suppl 77:S113-9 (2000)). A detailed understanding of the mechanismof reduced VEGF expression would provide a useful framework for newapproaches to improve diabetic outcomes following ischemic events.

Ischemia-Induced Neovascularization Occurs by Two Mechanisms:Angiogenesis and Vasculogenesis.

After the appropriate hypoxic signaling cascade is initiated,compensatory vascular growth in response to ischemic insult occurs bytwo different mechanisms (FIG. 1). In angiogenesis, mature residentendothelial cells proliferate and sprout new vessels from an existingvessel in response to an angiogenic stimulus. In a more recentlydescribed mechanism, termed vasculogenesis, circulating cells withcharacteristics of vascular stem cells (endothelial progenitor cells, orEPCs) are mobilized from the bone marrow in response to an ischemicevent, and then home specifically to ischemic vascular beds andcontribute to neovascularization (Asahara et al., “Isolation of PutativeProgenitor Endothelial Cells for Angiogenesis,” Science 275:964-7(1997); Shi et al., “Evidence for Circulating Bone Marrow-DerivedEndothelial Cells” Blood 92:362.7 (1998); Asahara at al., “Bone MarrowOrigin of Endothelial Progenitor Cells Responsible for PostnatalVasculogenesis in Physiological and Pathological Neovascularization,”Circ Res 85:2214 (1999); Isner et al., “Angiogenesis and Vasculogenesisas Therapeutic Strategies for Postnatal Neovascularization,” J ClinInvest 103:1231-6 (1999); Crosby et al., “Endothelial Cells ofHematopoietic Origin Make a Significant Contribution to Adult BloodVessel Formation,” Circ Res 87:728-30 (2000); Pelosi et al.,“Identification of the Hemangioblast in Postnatal Life,” Blood100:3203-8 (2002)).

Hypoxia-Inducible Factor-4 (HIF-1) is the Central Mediator of theHypoxia Response Including Subsequent Blood Vessel Growth.

The observation that ischemia regulates blood vessel growth has beenknown for many years, yet the responsible factor eluded identificationuntil 1992, when Semenza and colleagues described a hypoxia-responsivetranscription factor (HIF-1) which mediates erythropoietin geneupregulation (Semenza at al., “A Nuclear Factor Induced by Hypoxia viade Novo Protein Synthesis Binds to the Human Erythropoietin GeneEnhancer at a Site Required for Transcriptional Activation,” Mol CellBiol 12:5447-54 (1992); Semenza at al., “Hypoxia-Inducible NuclearFactors Bind to an Enhancer Element Located 3′ to the HumanErythropoietin Gene,” Proc Natl Acad Sci USA 88:5680-4 (1991)). HIF-1proved to be a novel transcription factor conserved in all metazoanphyla and is ubiquitously present in all cells examined thus far(Carmeliet et al., “Abnormal Blood Vessel Development and Lethality inEmbryos Lacking a Single VEGF Allele,” Nature 380:435-9 (1996)).Evidence for its involvement in angiogenesis stemmed from the initialobservation that VEGF was strongly upregulated by hypoxic conditions(Shweiki et al., “Vascular Endothelial Growth Factor Induced by HypoxiaMay Mediate Hypoxia-Initiated Angiogenesis,” Nature 359:843-5 (1992)).Soon thereafter, HIF-1 was shown to be the transcription factorresponsible for VEGF upregulation by hypoxia and hypoglycemia (Forsytheet al., “Activation of Vascular Endothelial Growth Factor, GeneTranscription by Hypoxia-Inducible Factor 1,” Mol Cell Biol 16:4604-13(1996)). It is now clear that HIF-regulated VEGF expression is essentialfor vascular development during both embryogenesis and postnatalneovascularization in physiologic and pathologic states (Carmeliet etal., “Abnormal Blood Vessel Development and Lethality in Embryos Lackinga Single VEGF Allele,” Nature 380:435.9 (1996); Carmeliet at al.,“Abnormal Blood Vessel Development and Lethality in Embryos Lacking aSingle VEGF Allele,” Nature 380:435-9 (1996); Iyer et al., “Cellular andDevelopmental Control of O2 Homeostasis by Hypoxia-Inducible Factor IAlpha,” Genes Dev 12:149-62 (1998)). HIF-1 consists of theoxygen-regulated HIF-1α subunit and the HIF-1β subunit, which is notregulated by oxygen. HIF-1 is now believed to be the mastertranscription factor directing the physiologic response to hypoxia byupregulating pathways essential for adaptation to ischemia, includingangiogenesis, vasculogenesis, erythropoiesis and glucose metabolism(FIG. 2).

Regulation of HIF-1α Transcriptional Activation.

The HIF-1 transcriptional complex is comprised of HIF-1α/β and more thanseven other factors that modulate gene transcription. The twopredominant functional components of this complex are HIF-1α andCBP/p300, which directly interact to transactivate gene expression.HIF-1α function is predominantly regulated by oxygen via proteinstabilization and post-translational modification. Recent reportsdemonstrate that HIF-1α is activated by phosphorylation in vitro,enhancing HIF-mediated gene expression (Richard et al., “p42/p44Mitogen-Activated Protein Kinases Phosphorylate Hypoxia-Inducible FactorI alpha (HIF-1 alpha) and Enhance the Transcriptional activity ofHIF-1,” J Biol Chem 274:32631-7 (1999)). Whether this modificationresults in a direct stimulation of the transactivation function ofHIF-1α itself or facilitates recruitment of co-activators is not clear(Richard t al, “p42/p44 Mitogen-Activated Protein Kinases PhosphorylateHypoxia-Inducible Factor I alpha (HIF-1 alpha) and Enhance theTranscriptional activity of HIF-1,” J Biol Chem 274:32631-7 (1999); Sanget al., “Signaling Up-Regulates the Activity of Hypoxia-InducibleFactors by Its Effects on p300,” J Biol Chem 278:14013-9 (2003)).

It has also been recently demonstrated that CBP/p300 also undergoesphosphorylation in vitro, enhancing its ability to function as atranscriptional activator in association with HIF-1α (Sang et al.,“Signaling Up-Regulates the Activity of Hypoxia-Inducible Factors by ItsEffects on p300,” J Biol Chem 278:14013-9 (2003)). Thus, cellular statesthat promote phosphorylation of these two factors likely increasehypoxia-induced gene expression, while those that favordephosphorylation have the opposite effect. Although HIF-1 mediated geneexpression is essential for both angiogenesis and vasculogenesis, therole of its regulation in diabetic states has not been previouslyexamined.

Both Angiogenesis and Vasculogenesis are Modulated by VEGF.

It is well known that angiogenesis is mediated by VEGF and thismechanism has been extensively investigated (Ferrara et al., “TheBiology of VEGF and its Receptors,” Nat Med 9:669-76 (2003)). Recently,VEGF has also been implicated in regulation of vasculogenesis (FIG. 2).Ischemia is a potent mobilizer of endothelial progenitor cells from thebone marrow. This appears to be mediated through VEGF signaling, as EPCsexpress both VEGF receptor I and 2 on their cell surface (Asahara etal., “VEGF Contributes to Postnatal Neovascularization by MobilizingBone Marrow-Derived Endothelial Progenitor Cells,” Embo J 18:3964-72(1999); Takahashi et al., “Ischemia- and Cytokine-Induced Mobilizationof Bone Marrow-Derived Endothelial Progenitor Cells forNeovascularization,” Nat Med 5:434-8 (1999); Kalka et al., “VascularEndothelial Growth Factor(165) Gene Transfer Augments CirculatingEndothelial Progenitor Cells in Human Subjects,” Circ Res 86:1198-202(2000); Gill at al., “Vascular Trauma Induces Rapid but TransientMobilization of VEGFR2(+)AC133(+) Endothelial Precursor Cells,” Circ Res88:167-74 (2001); Hattori et al., “Vascular Endothelial Growth Factorand Angiopoietin-1 Stimulate Postnatal Hematopoiesis by Recruitment ofVasculogenic and Hematopoietic Stem Cells,” J Exp Med 193:1005-14(2001)). Given that VEGF production is impaired in diabetes mellitus, itseems likely that various aspects of vasculogenesis, including EPCmobilization, may also be impaired. Indeed, recent evidence hasdemonstrated that the incorporation of these vascular progenitors intoblood vessels is decreased in diabetic states.

VEGF Expression May be Regulated in a Tissue-Specific Manner.

It also clear that various tissues and organs in diabetic patientsexhibit different pathologies. The retina is often characterized byexcessive angiogenesis, while skin, muscle, and nerves in diabeticpatients suffer from a paucity of new vessel formation. Similarly,diabetic retinopathy has been characterized by increased levels ofocular VEGF levels, (Aiello et al., “Vascular Endothelial Growth Factorin Ocular Fluid of Patients with Diabetic Retinopathy and Other RetinalDisorders,” N Engl J Med 331:1480-7 (1994); Adamis t al., “IncreasedVascular Endothelial Growth Factor Levels in the Vitreous of Eyes withProliferative Diabetic Retinopathy,” Am J Ophthalmol 118:445-50 (1994)),while impaired wound healing has been characterized by severelydecreased levels of VEGF (Frank et al., “Regulation of VascularEndothelial Growth Factor Expression in Cultured Keratinocytes.Implications for Normal and Impaired Wound Healing,” J Biol Chem270:12607-13 (1995); Peters et al., “Vascular Endothelial Growth FactorReceptor Expression During Embryogenesis and Tissue Repair Suggests aRole in Endothelial Differentiation and Blood Vessel Growth,” Proc NatlAcad Sci USA 90:8915-9 (1993); Silhi, N., “Diabetes and Wound Healing,”J Wound Care 7:47-51 (1998); Brown, L. F., “Expression of VascularPermeability Factor (Vascular Endothelial Growth Factor) by EpidermalKeratinocytes During Wound Healing,” J Exp Med 176:1375-9 (1992); Nissenet al., “Vascular Endothelial Growth Factor Mediates Angiogenic ActivityDuring the Proliferative Phase of Wound Healing.” Am J Pathol152:1445-52 (1998)). This so-called “diabetic paradox,” by which thediabetic phenotype exhibits both excessive and impaired new blood vesselformation in different tissues, leads to different types ofcomplications. It is believed this phenomenon represents a cell- andtissue-specific difference in the transcriptional regulation of VEGF.

Hyperglycemia Results in Specific Impairments of Cellular FunctionThrough Overproduction of Reactive Oxygen Species: a Potential Link toVEGF.

The cellular mechanism that accounts for impaired hypoxia-induced VEGFand SDF-1 expression has not yet been determined. Recently, thebiochemical basis for hyperglycemia-induced cellular damage wasdescribed, demonstrating that many of the effects of high glucose aremediated through four specific cellular pathways (FIG. 3) (Brownlee,“Biochemistry and Molecular Cell Biology of Diabetic Complications,”Nature 414:813-20 (2001); Nishikawa et al. “Normalizing MitochondrialSuperoxide Production Blocks Three Pathways of Hyperglycaemic Damage,”Nature 404:787-90 (2000)). Intracellular elevations in glucose increaseflux of metabolites through glycolysis and the Kreb's cycle, resultingin overproduction of ROS by the mitochondria. Overproduction of ROSinhibits GAPDH activity, resulting in accumulation of early glucosemetabolites in the initial phases of glycolysis. The abundance of thesemetabolites and their inability to progress through glycolysis causesshunting of these intermediates into alternative pathways of glucoseutilization (polyol pathway, hexosamine pathway, protein kinase Cpathway, and AGE pathway, FIG. 3). Accumulation of end products in eachof these pathways leads to specific changes in cellular function,including gene expression (Nissen et al., “Vascular Endothelial GrowthFactor Mediates Angiogenic Activity During the Proliferative Phase ofWound Healing” Am J Pathol 152:1445-52 (1998)), and are implicated inthe pathophysiology of diabetic complications (Brownlee, “Biochemistryand Molecular Cell Biology of Diabetic Complications,” Nature 414:813-20(2001)). Indeed, specific blockade of one, several, or all of thesepathways has been shown to prevent diabetic complications in an animalmodel, including those complications that result from ischemic injury(Hammes at al., “Benfotiamine Blocks Three Major Pathways ofHyperglycemic Damage and Prevents Experimental Diabetic Retinopathy,”Nat Med 9:294-9 (2003); Obrosova et al., “Aldose Reductase InhibitorFidarestat Prevents Retinal Oxidative Stress and Vascular EndothelialGrowth Factor Overexpression in Streptozotocin-Diabetic Rats,” Diabetes52:864-71 (2003)).

Hyperglycemia-induced reactive oxygen species also impair the ability ofHIF-1α to mediate appropriate upregulation of VEGF and the chemokineSDF-1 that are required for neovascularization in ischemic settings.This impairment also affects hypoxia-specific functions of vasculareffector cells. This results in impaired angiogenesis, vasculogenesis,and diminished tissue survival in diabetic states. Increased free fattyacid flux has been shown to increase ROS by identical mechanisms (Du tal., “Insulin Resistance Causes Proatherogenic Changes in ArterialEndothelium by Increasing Fatty Acid Oxidation-Induced SuperoxideProduction” J. Clin. Invest. in press).

The present invention is directed to treating or preventing thepathologic sequelae of acute hyperglycemia and/or increased fatty acidflux in a subject, thus, preventing metabolite-induced reactiveoxygen-species mediated injury.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method of treating orpreventing pathologic sequelae of acute hyperglycemia and/or increasedfatty acid flux in non-diabetic subjects, metabolic syndrome/insulinresistance subjects, impaired fasting glucose subjects, impaired glucosetolerance subjects, and diabetic subjects. This method involvesadministering an ROS inhibitor to the subject under conditions effectiveto treat or prevent pathologic sequelae of acute hyperglycemia and/orincreased fatty acid flux in the subject.

Another aspect of the present invention relates to a method of promotingneovascularization in a subject prone to hyperglycemia or increasedfatty acid flux. This method involves administering an ROS inhibitor tothe subject under conditions effective to promote neovascularization inthe subject.

A further aspect of the present invention pertains to a method ofinhibiting oxidation or excessive release of free fatty acids in asubject. This method involves administering to the subject certaincompounds under conditions effective to inhibit excessive release offree fatty acids in the subject. These compounds includethiazolidinedione nicotinic acid, etomoxir, and ranolazine.

A further aspect of the present invention is directed to a method ofidentifying compounds suitable for treatment or prevention ofROS-mediated injury. This method involves providing a diabetic animalmodel and inducing diabetes in the animal model. A compound to be testedis then administered to the animal model. Compounds which achieverecovery of local oxygen tension, blood flow, increase in vesseldensity, and tissue survival in the animal model as therapeuticcandidates for treating or preventing ROS-mediated injury are thenrecovered.

The present invention provides a means of restoring deficientangiogenesis in response to ischemia in patients with disorders ofglucose and fatty acid metabolism. This would drastically reduce therate of lower limb amputation, and reduce the extent of cardiac andbrain damage due to heart attacks and strokes. In addition, it wouldresult in healing of intractable diabetic foot ulcers, a major clinicalproblem for which there is currently no available effective medicaltreatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of angiogenesis and vasculogenesis.

FIG. 2 shows the central role of HIF and VEGF in the ischemic response.

FIG. 3 shows pathways of hyperglycemic damage.

FIG. 4 shows an overall experimental plan.

FIG. 5 shows a murine model of graded cutaneous ischemia. Regions A, B,and C reflect increasingly ischemic tissue regions, as measured bydirect tissue oxygen tension at reference points p1 (27 mm Hg)-p5 (6 mmHg).

FIG. 6 shows tissue survival in diabetic mice.

FIG. 7 shows oxygen tension measurements in ischemic tissue from leastischemic (p1) to most ischemic (p5) compared to normal skin oxygentension (NL Skin).

FIG. 8 shows the number of blood vessels identified by CD31 staining inareas A, B, and C of ischemic flaps.

FIG. 9 shows oxygen tension measurements post-operatively in wild typeand MnSOD transgenic mice with streptozotocin-induced diabetes.

FIG. 10 shows JC-1 staining of C2Cl2 myoblasts cultivated in normalglucose (5 mM), as well as acute and chronic high glucose (25 mM).

FIG. 11 shows VEGF mRNA in high and low glucose in response to hypoxia.

FIG. 12 shows VEGF mRNA half life in cells cultivated in normal glucose(●) or high glucose (∘) conditions.

FIG. 13 shows VEGF promoter activity in C2Cl2 myoblasts in normal (5 mM,grey) and high glucose (25 mM, black) after hypoxic stimulus.

FIG. 14 shows HIF-Id is preferentially glycosylated in high glucoseconditions (HG, 25 mM) compared to normal glucose (NG, 5 mM).

FIGS. 15 A-B show high glucose (30 mM) impairs the association betweenp300 and PPARγ. This effect was abolished by inhibiting GFAT.

FIGS. 16A-C show pathways of cellular damage resulting from reactiveoxygen species can be selectively targeted and prevented.

FIGS. 17A-B show fibroblasts from diabetic mice do not demonstrate anormal hypoxia-induced increase in migration seen in non-diabetic cells(p<0.05).

FIGS. 18A-B show fibroblasts from diabetic mice produce more proMMP-9,but not active MMP-9 (p<0.001).

FIGS. 19A-D show EPCs from Type II diabetics proliferate less duringexpansion (A) which inversely correlated with HbA1c levels (B). FewerEPC clusters formed in culture (C), which was also inversely correlatedto the total number of years with diabetes.

FIGS. 20A-C show EPCs from Type II diabetic patients are impaired intheir ability to adhere, migrate, and proliferate in response to hypoxicstimuli (&=p<0.001, &&=p<0.05).

FIG. 21 shows the effect of deferoxamine on hyperglycemia-induced ROS.

FIG. 22 shows the free intracellular iron measurement in bovine aorticendothelial cells after infection with UCP-1, Mn-SOD or empty adenoviralvectors and subsequent treatment with 5 mM or 30 mM glucose. The x-axisshows the different treatments. The y-axis shows fluorescence unitsindicating the amount of free iron.

FIGS. 23A-C show the free intracellular iron measurement in bovineaortic endothelial cells after incubation with 5 mM or 30 mM glucose(FIGS. 23A and 238, respectively) or 30 mM glucose plus 100 μMdeferoxamine (FIG. 23C) for 24 hours. Detection of free iron wasaccomplished by visualizing the fluorescent marker fura-2 AM.

FIGS. 24A-D show, respectively, DNA strand breakage in aorticendothelial cells after incubation with 5 mM or 30 mM glucose or 30 mMglucose plus 100 μM deferoxamine for 7 days.

FIG. 25 shows PARP activity in aortic endothelial cells after incubationwith 5 mM or 30 mM glucose, 30 mM glucose plus 100 μM deferoxamine, or30 mM glucose plus 100 μM DMSO for 6 days. ³H NAD incorporation was usedto assess PARP activity. The x-axis shows the different treatments. They-axis shows the PARP activity as measured in pmol/mg protein.

FIG. 26 shows prostacyclin synthase activity in aortic endothelial cellsafter 24 hour incubation with 5 mM or 30 mM glucose, or 30 mM glucoseplus 100 μM deferoxamine. The x-axis shows the different treatments. They-axis shows the prostacyclin synthase activity expressed asconcentration of the prostacyclin synthase product PGF-1α.

FIG. 27 shows prostacyclin synthase activity in aortas of diabetic andcontrol mice after daily deferoxamine injections for 7 days. The x-axisshows the different treatments. The y-axis shows the prostacyclinsynthase activity as measured by the concentration of the prostacyclinsynthase product PGF-1α.

FIG. 28 shows eNOS activity in aortic endothelial cells after incubationwith 5 mM or 30 mM glucose or 30 mM glucose plus 100 μM deferoxamine for24 hours. The x-axis shows the different treatments. The y-axis showsthe eNOS activity as a function of ³H-citrulline generated per minuteper 10⁵ cells.

FIG. 29 shows eNOS activity in aortas of diabetic and control mice afterdaily deferoxamine injections for 7 days. The x-axis shows the differenttreatments. The y-axis shows the eNOS activity as a function of³H-citrulline generated per minute per mg of protein.

FIGS. 30A-F show a diabetes-induced defect in mouse angiogenic responseto ischemia. FIGS. 30A-B show oxygenation levels in non-diabetic anddiabetic mice, respectively. P1-P4 on the y-axis designate adjacentquadrants of the ischemic skin flap starting closest to the site ofattachment to the animal, i.e. P1, and proceeding distally to P4. FIGS.30C-D show mobilization of bone marrow-derived endothelial cells inresponse to ischemia. Flk-1 on the y-axis is a marker for ischemicbone-marrow-derived endothelial precursor cells. CDI lb on the x-axis isa general marker for bone marrow-derived endothelial precursor cells.FIGS. 30E-F show the amount of capillary formation in non-diabetic anddiabetic mice, respectively. NI on the y-axis represents capillarydensity of a non-ischemic control. Area C on the y-axis represents thecapillary density in an ischemic skin flap after 7 days.

FIGS. 31A-C show that deferoxamine treatment corrects thediabetes-induced defect in mouse angiogenic response to ischemia. Thetreatment groups were wild-type mice (WT), streptozotocin-induceddiabetic mice (shown as STZ in FIG. 31 C, and DM in FIGS. 31 A-B), anddeferoxamine-treated streptozotocin-induced diabetic mice (shown asSTZ+deferox in FIG. 31 C, and DM+DEF in FIGS. 31A-B).

FIG. 32 shows CD 31 positive blood vessel counts in wild-type mice (WTC), wild-type mice treated with deferoxamine (WT Def C),streptozotocin-induced diabetic mice (STZ C), and streptozotocin-induceddiabetic mice treated with deferoxamine (STZ Def C). The y-axis showsthe CD 31 positive blood vessel counts per hpf (high powered field).

FIGS. 33A-B show that deferoxamine treatment corrects thediabetes-induced defect in mouse angiogenic response to ischemia.Diabetes was induced in the mice by streptozotocin (abbreviated STZ inFIG. 33 A-B). The mouse shown in FIG. 33A was treated with vehiclealone, while the diabetic mouse shown in FIG. 33B was treated withdeferoxamine (labeled as STZ Deferoxamine in FIG. 33 B).

FIGS. 34 A,C, and E show that diabetes-induces defects in mouseangiogenic response to ischemia. FIGS. 34B,D, and F that these defectsare corrected by treatment with deferoxamine. FIGS. 34 A-B showoxygenation levels in streptozotocin-induced diabetic (FIG. 33 A) anddeferoxamine-treated streptozotocin-induced diabetic mice (FIG. 33 B)respectively. P1-P4 on the y-axis designate adjacent quadrants of theischemic flap starting closest to the site of attachment to the animal,i.e., P1, and proceeding distally to P4. FIGS. 34 C-D show mobilizationof bone marrow derived endothelial cells in response to ischemia. Flk-1on the y-axis is a marker for bone-marrow derived endothelial precursorcells. CD11b on the x axis is a general marker for bone-marrow derivedcells of the myeloid, macrophage, and granulocytic lines. FIGS. 34 E-Fshow the amount of capillary formation in vehicle-treatedstreptozotocin-induced diabetic and deferoxamine-treatedstreptozotocin-induced diabetic mice respectively. NI on the y-axisrepresents capillary density of a non-ischemic control. Area C on they-axis represents the capillary density at the most distal third of anischemic skin flap alter 7 days.

FIG. 35 is a bar chart showing EPC mobilization for control, STZ, andSTZ-deferoxamine mice. EPC mobilization was determined at day 7postischemic insult. Diabetes resulted in a 3-fold decrease in EPCmobilization. Deferoxamine restores ischemia specific EPC mobilizationas compared to untreated STZ mice (p<0.05).

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a method of treating orpreventing pathologic effects of acute increases in hyperglycemia and/oracute increases of fatty acid flux in non-diabetic subjects, metabolicsyndrome/insulin resistance subjects, impaired fasting glucose subjects,impaired glucose tolerance subjects, and diabetic subjects. This methodinvolves administering an ROS inhibitor to the subject under conditionseffective to treat or prevent pathologic effects of acute increases inhyperglycemia and/or acute increases in fatty acid flux in the subject.

As noted above, in this aspect of the present invention, the claimedmethod can be applied to non-diabetic subjects, metabolicsyndrome/Insulin resistance subjects, impaired fasting glucose subjects,impaired glucose tolerance subjects, and diabetic subjects. In eachcase, the subject has a base line level of hyperglycemia and/or fattyacid flux. The present invention is directed to the prevention ortreatment of pathologic conditions in subjects whose base line levels ofhyperglycemia and/or fatty acid flux undergo a rapid and relativelyshort-term (i.e. acute) increase.

Subjects where acute increases in hyperglycemia and/or acute increasesof fatty acid flux take place may be suffering from any of the followingconditions: diabetes-specific microvascular pathology in the retina(i.e. diabetic retinopathy), renal glomerulus (i.e. diabeticnephropathy), peripheral nerve (i.e. diabetic neuropathy), acceleratedatherosclerotic macrovascular disease affecting arteries that supply theheart, brain, and lower extremities (i.e. diabetic macrovasulardisease), or nonalcoholic fatty liver disease (“NAFLD”) which includes awide spectrum of liver injury ranging from simple steatosis tosteatohepatitis (“NASH”), fibrosis, and cirrhosis. The pathologic effectof acute increases in hyperglycemia and/or acute increases of fatty acidflux may also be prevented or treated where the subject has a criticalcare illness, an acute myocardial infarction, an acute stroke, or whohas undergone arterial bypass or general surgery.

Acute increases in hyperglycemia and/or acute increases of fatty acidflux impairs mobilization of vascular endothelial cell precursors fromthe bone marrow. This may take the form of impairing mobilization ofvascular endothelial cell precursors from the bone marrow, impairingHIF-1α- and SDF-1-mediated upregulation of vascular endothelial growthfact, and/or ROS-mediated injury which inhibits neovascularization. Thesubject can also have an ischemic condition which includes coronaryartery disease, peripheral vascular disease, cerebral vascular disease,non-healing foot ulcers, or a wound (acute or chronic).

The ROS generation by hyperglycemia or increased fatty acid flux takesplace in the mitochondria. The most common ROS are hydrogen peroxide(H₂O₂), hydroxyl radicals (OH.), lipid peroxy radicals (LOO⁻), andperoxynitrites (ONOO⁻).

H₂O₂ is relatively stable and can diffuse through membranes. In mostcells, H₂O₂ is detoxified by enzymatic reduction to H₂O and O₂. Inmitochondria, the enzyme glutathione peroxidase is primarily responsiblefor this reaction. In the cytosol and peroxisomes, both glutathioneperoxidase and the enzyme catalyse mediate this reaction. However, inthe presence of free d-block transition metals, such as iron, theoxidized form of the metal is thought to react with superoxide,producing the oxidized from of the metal and molecular oxygen (O₂). Thereduced metal then reacts with H₂O₂ to regenerate the initial oxidizedmetal, hydroxyl ions (OH⁺) and hydroxyl radicals (OH.). It is importantto note, however, that this chemistry is still far from beingunderstood.

It is well known that iron and other d-block transition metals canfunction as free-radical catalysts, potentially generating toxic speciessuch as hydroxyl radicals. Transition metals are the large block ofelements in the Periodic Chart that have group numerical designationsending with B, such as IB, IIB, IIIB, and so on. They are the four rowsof ten elements located in the heart of the chart. They are also theelements whose final electron enter the d orbital (called d-blockmetals). All first row d-block metals (except zinc) have unpairedelectrons (Sc, Ti, V, Cr, Mn, fe, Co, Ni, and Cu) which removes spinrestrictions and allows then to function in free radical catalysis, bothas elements bound at the active site of enzymes, and free in solution.

Iron has been the focus of many chemical studies because this chemistrywas first demonstrated by H. J. H. Fenton in 1876 using unchelatedFe²⁺/H₂O₂ mixture in aqueous solution. To distinguish between the iron(II) and iron (III) combinations, the convention is to use Fenton-likereagent for the Fe³⁺/H₂O₂ mixture and restrict the use of Fenton'sreagent to denote the Fe²⁺/H₂O₂ The Fenton-like reagent is also capableof oxidizing organic substrates, but it is somewhat less reactive thanFenton's reagent. As iron(III) can be produced in applications ofFenton's reagent, Fenton chemistry and Fenton-like chemistry often occursimultaneously.

Fenton reagent chemistry is still far from being fully understood, andFenton-like reagent chemistry even less well understood. Numerousreaction mechanisms have been proposed for Fenton reagent chemistrybased on different active intermediates such as OH. and OOH. radicalsand high-valent iron species. Haber and Weiss's OH. radical mechanisms(citation) is probably the most popular candidate for the Fentonreaction:

Fe²⁺+H₂O₂→Fe³⁺+OH⁻+OH⁻

A popular alternative mechanistic candidate is that first suggested byBray and Gorin (citation), in which the ferryl ion, [Fe^(IV) O]²⁺, issupposed to be the active intermediate:

Fe²⁺H₂O₂→[Fe^(IV)O]²⁺+H₂O

In addition to reducing hydrogen peroxide, ferrous iron can also reactwith alkyl hydroperoxides, to produce alkoxyl radicals. These alkoxylradicals can then initiate the oxidation of polyunsaturated lipids by aflee radical chain reaction (citation).

Despite its appearance in numerous biochemistry textbooks, thebiological significance of Fenton chemistry has been questioned by manyfree radical chemists in part because the rate constants for thereaction of reduced metals and their complexes with H₂O₂ are not rapid,and their in vivo metal ligands are unknown (citation). However, it hasbeen shown recently that HCO₃ ⁻ and CO₂ greatly accelerate the rate ofH₂O₂ reduction by Mn²⁺ and other transition metals (citation). It isprobable that many of the proposed mechanisms compete with each other incomplex and unpredictable ways, depending on the reaction conditions,such as the metal ligands, their valence, the solvent, the pH and theorganic substrate to be oxidized (citation)

The ROS inhibitor can be alpha lipoic acid, a superoxide dismutasemimetic, or a catalase mimetic. The superoxide dismutase mimetic or thecatalase mimetic can be MnTBAP (Mn(III)tetrakis(4-benzoic acid)porphyrinchloride)(produced by Calbiochem), ZnTBAP (Zn(III)tetrakis(4-benzoicacid)porphyrin chloride), SC-55858 (manganese (11) dichloro(2R,3R,8R,9R-bis-cyclohexano-1,4,7,10,13-pentaazacyclopentadecane)]Euk-134 (3,3′-methoxysalenMn(III)) (produced by Eukarion).

Alternatively, the ROS inhibitor can be an iron chelator or acomposition comprising a mixture of iron chelators. Chelators are smallmolecules that bind very tightly to metal ions. The key property sharedby all chelators is that the reactivity of the metal ion bound to thechelator is greatly reduced, although in some cases and under certainconditions, chelator metal-complexes themselves can generate reactiveoxygen free radicals. Clinically useful chelators must be highlyspecific for one d-block transition metal such as iron. Chelators whichare non-specific are highly toxic.

The basic property of a chelator consists in having the ability offorming a heterocyclic ring structure with a metal ion as the closingmember. The chelator must possess two or more functional groups(ligands) with atoms which can donate a pair of electrons for theformation of a bond with the metal ion. Donor atoms are usually N, O andS, which can function either as members of an acidic group such as:—COOH, OH (phenolic, enolic), —SH, —NH═O, —NOH in which case the protonis displaced by the metal ion, or as lone pair of electron donors (Lewisbase) such as —C═O, —NH₂, —O—R, —OH (alcoholic), —S-thioether, asdescribed in Current Medicinal Chemistry, 2004, 11, 2161-2183,incorporated herein by reference in its entirety.

Ideally, tight binding of iron to a chelator should completely inhibitits ability to function as a free-radical catalyst. Iron chelators areclassified according to the stoichiometry of binding with iron. Ironions have six electrochemical coordination sites. Thus, a chelatormolecule that binds to all six sites in a 1:1 ratio is called‘hexidentate.” A chelator molecule that binds to only two of the sixsites is called “bidentate,’ and chelators that bind to three of the sixsites are called “tridentate.’ In theory, three molecules of a bidentatechelator should reduce free iron reactivity as completely as onemolecule of a hexidentate chelator. However, with bidentate ironchelators, formation of fee-radical catalyzing partial reductionproducts often occurs. Practically, this means that a large chemicalexcess of such chelators is needed in order to avoid the formation ofthese reactive chelator iron complexes.

Iron chelators can be classified using a number of criteria such astheir origin (synthetic versus biologically produced molecules), theirinteraction with solvents such as water (hydrophobic versus hydrophilic)or their stoichiometric interaction (bidentate versus hexadentate).

A general structure of an effective iron chelator comprises the genericstructure R-L-C-M and all the combinations thereof.

For example, C can represent the iron-chelating moiety bi, tri orhexidentate characterized by selective iron-binding affinity and avidityas described in Current Medicinal Chemistry, 2004, 11, 2161-2183. Thecombination C-M can represent a bi-functional drug structure containingan iron-chelating moiety C bound to a masking group M which could be anelectron-donor atom. Intracellular hydroxyl radicals OH. can be reducedby the electrons of M, cleave M from C which, once unmasked, can bindfree iron ions.

The combination R—C can represent the iron chelating moiety C bound to aback bone side-chain R, wherein R can be H, a linear aliphatic chainstructure, or an alifatic chain including aromatic, alifatic and/orheteroaromatic rings. Because the relative potency of chelators appearsto be related to the hydrophilicity of the molecule, the chemicalstructure of R can facilitate or hinder the penetration of the chelatorsin target cells and/or target cell compartments. Finally, thecombination R-L-C can represent the iron chelating moiety C bound to aside-chain R through a linker L. The linker L can facilitate a rapidcellular intake and delay the cellular exit of C as described in J. Am.Chem. Soc. 2002, 124, 12666-12667, incorporated herein by reference inits entirety. For example, R-L-C can represent a prohydrophilic drug (apro-drug). L can be an ester bond, R an ester moiety and C an ironchelating moiety. Upon entrance in the cell, R-L-C can turn highlyhydrophilic upon esterase-mediated hydrolysis of the lipophilic moietyR. Thus, L is hydrolyzed, R is chemically detached from the molecule andthe more hydrophilic C is retained inside the cell where it can performits chelating function.

Other general structures of effective iron chelators comprise the familyof 3,5-diphenyl-1,2,4-triazoles of the formula I described in U.S. Pat.No. 6,465,504 incorporated herein by reference in its entirety.

Of the iron chelators, deferoxamine or DFO may be the most important,because it is FDA-approved for treatment of iron excess in thallasemia.

When deferoxamine is employed, a patient (e.g., a patient with an acutemyocardial infarction) can be treated with intramuscular injections of1,000 to 10,000 mg of deferoxamine or with intravenous injections of 100to 10,000 mg of deferoxamine. Such patients can be treated within 24hours of symptoms by intravenous injection of deferoxamine in liquidform at a concentration between 100 to 10,000 mg/liter of deferoxamine.Deferoxamine can also be administered together with DFP, ICL-670, a poly(ADP-ribose) polymerase inhibitor, and a glucagon-like peptide-1fragment that prevents hyperglycemia-induced ROS production, forexample, GLP-1 (9-36 amide), and GLP-1 9-37). Alternatively,deferoxamine can be administered together with a poly (ADP-ribose)polymerase inhibitor including, but not limited to, nicotinamide,3-aminobenzamide, P134 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-NN-dimethylacetamide), and mixtures thereof.

While deferoxamine can provide life-saving treatment for patients iniron overload situations, numerous deferoxamine derivatives can also beemployed. Aliphatic, aromatic, succinic, and methylsulphonic analogs ofDFO have been synthesized to enhance the lipophilicity of DFO (Ihnat etal., “Solution Equilibria of Deferoxamine Amides,” J. Pharm Sci.91:1733-1741 (2002), which is hereby incorporated by reference in itsentirety). Specifically, these derivatives includeformamide-deferoxamine, acetamide-deferoxamine, propylamidedeferoxamine, butylamide-deferoxamine, benzoylamide-deferoxaminesuccinamide-deferoxamine, and methylsulfonamide-deferoxamine.Hydroxylethyl starch (HES)-deferoxamine has been synthesized which wasshown to have a greater plasma half-life than deferoxamine (Pedchenko etal., “Desferrioxamine Suppresses Experimental Allergic EncephalomyelitisInduced by MBP in SJL mice,” J. Neuroimmunol. 84:188-197 (1998), whichis hereby incorporated by reference in its entirety). Anaminooxyacetyl-ferrioxamine has also been prepared allowing for sitespecific conjugation to antibodies (Pochon et al., “A novel Derivativeof the Chelon Desferrioxamine for Site-specific Conjugation toAntibodies,” Int. J. Cancer. 43:1188-1194 (1989), which is herebyincorporated by reference in its entirety. Fluorescent deferoxaminederivatives have also been synthesized for free iron measurements in arange of biological experimental conditions (Al-Mehdi et al.,“Depolarization-associated iron release with abrupt reduction inpulmonary endothelial shear stress in situ,” Antioxid. Redox Signal.2:335-345 (2000), which is hereby incorporated by reference in itsentirety.

Other suitable iron chelators include those set forth in Table 2:

PHARMACOLOGY Name Formula Chem. structure MW Dent Route DFO 4-[3,5- bis-[hy- droxy- phenyl]- 1,2,4- triazol- 1-yl]- benzoic acid

560    6 par- ent- al HBED N,N′- bis(o- hy- droxy- benzyl) ethyl- ene-damine- N,N′- diacetic acid

388    6 oral/ par- ent- al PIH pyri- doxal isanico- tinoyl hydra- zone

262    3 oral DFT 4′-hy- droxy- (S)- desaza des- methyl- desferri-thiocin; (S)-4,5- di- hydro-2- (2,4- dihydro- phenyl)- 4- thiazo- fecar-boxylic acid

238    3 oral DFP (L1) 1,2- di- methyl- 3-hy- droxy- pyridin- 4-one

139    2 oral S- hydroxy- — 250.000 6 i.v. DFO ethyl- starch- bound-deferox- amine ICL- 670 4-[3,5- bis- (hy- droxy- phenyl)- 1,2,4-triazol- 1-yl]- benzoic acid

373    3 oral GT56- 252 4,5-di- hydro-2- (2,4- dihy- droxl- phenyl)- 4-methyl- thiazole- 4(S)- car- boxylic acid

252    3 oral

HEBED is a synthetic chelator that appears to have higher efficacy thanDFO, and fewer adverse effects. However, in primate studies, it stillhad to be administered by subcutaneous infusion (Chaston, et. al., “IronChelators for the Treatment of Iron Overload Disease: RelationshipBetween Structure, Redox Activity, and Toxicity” Am J Hematol.73:200-210 (2003), which is hereby incorporated by reference in itsentirety.

PIH is an orally active, triedentate chelator which crosses membranesmuch better than does DFO. PCHI (i.e. analogues of2-pyridylcarboxaldehyde isonicotinoyl hydrazone) compounds (which arenot shown in Table 2) are substantially similar to PIH. This class ofchelators can also access mitochondrial iron pools, making it apotential drug for the rare genetic disease Friedrich's Ataxia (causedby a mutation in the mitochondrial iron-sulfur complex chaperonefrataxin).

Like HBED, DFT and GT56-252 are both second generation hydroxypyridonesthat are in preclinical or phase I trials.

DFP or Deferipone, is approved for clinical use in Europe under thetrade name Ferriprox. It is a bidentate chelator that is administeredorally. However, the efficacy and toxicity of the drug are stillcontroversial. Combined use of DFO and DFP has been proposed.

S-DFO is a starch-bound DFO derivative that has a longer half-life afterintravenous administration.

ICL-670 is a tridentate chelator of the triazole family currently inphase II trials. It is orally available and is administered once a day(Hershko, C., et al., Blood 97:1115-1122 (2001), which is herebyincorporated by reference in its entirety).

Another class of iron chelator is the biomimetic class (Meijier, M M, etal. “Synthesis and Evaluation of Iron Chelators with Masked HydrophilicMoieties” J. Amer. Chem. Soc. 124:1266-1267 (2002), which is herebyincorporated by reference in its entirety). These molecules are modifiedanalogues of such naturally produced chelators as DFO and ferrichrome.The analogues allow attachment of lipophilic moieties (e.g.,acetoxymethyl ester) which greatly enhance passage through membranes.The lipophilic moieties are then cleaved intracellularly by endogenousesterases, converting the chelators back into hydrophilic moleculeswhich cannot leak out of the cell. These compounds appear to be highlyeffective, and reduce free-iron mediated oxidative damage much moreefficiently than does DFO.

Lastly, a number of compounds developed as inhibitors of advancedglycation endproduct (AGE) formation and/or degradation and tested inanimal models of diabetic complications appear to act via chelation(Price, D L, et al., JBC 276:48967-72 (2001), which is herebyincorporated by reference in its entirety). These include (in order fromweakest to strongest copper chelation): aminoguanidine and pyridoxamine;carnosine, phenazinediamine, OPB-9195, and tenilsetam. The so-calledAGE-breakers, phenacylthiazoloum and phenacyldimethythiazolium bromide,and their hydrolysis products, were among the most potent inhibitors ofcopper-catalyzed autoxidation of ascorbate. Aminoguanidine has beenthrough Phase II/III trials, pyridoxamine has been through Phase IItrials, and the AGE breakers are currently in Phase II trials.

The inhibitors can be administered orally, parenterally, transdermally,subcutaneously, intravenously, intramuscularly, intraperitoneally, byintraversal instillation, intracularly, intranasally, intraarterially,intralesionally, or by application to mucous membranes, such as that ofthe nose, throat, and bronchial tubes. The inhibitors can beadministered alone or with a pharmaceutically acceptable salt, carrier,excipient, or stabilizer, and can be in solid or liquid form, including,for example, tablets, capsules, powders, solutions, suspensions, oremulsions.

The solid unit dosage forms can be of the conventional type. The solidform can be a capsule, such as an ordinary gelatin type containing theinhibitors of the present invention and a carrier, for example,lubricants and inert fillers such as lactose, sucrose, or cornstarch. Inanother embodiment, the inhibitors are tableted with conventional tabletbases such as lactose, sucrose, or cornstarch in combination withbinders like acacia, cornstarch, or gelatin, disintegrating agents suchas, cornstarch, potato starch, or alginic acid, and a lubricant likestearic acid or magnesium stearate.

In another aspect, the inhibitors of the present invention may be orallyadministered, for example, with an inert diluent, or with an assimilableedible carrier, or they may be enclosed in hard or soft shell capsules,or they may be compressed into tablets, or they may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the inhibitors of the present invention may be incorporated withexcipients and used in the form of tablets, capsules, elixirs,suspensions, syrups, and the like. In one aspect, such formulationsshould contain at least 0.1% of the inhibitors of the present invention.The percentage of the inhibitors in the formulations of the presentinvention may, of course, be varied and may conveniently be betweenabout 2% to about 60% of the weight of the unit. The amount ofinhibitors in the formulations of the present invention is such that asuitable dosage will be obtained. As one example, formulations accordingto the present invention are prepared so that an oral dosage unitcontains between about 1 and 250 mg of the inhibitors.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor.

As described above, in one aspect of the present invention, theformulations containing the inhibitors may be administered parenterally.Solutions or suspensions of the inhibitors can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof in oils. Illustrative oils are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, or mineral oil. In general, water, saline, aqueousdextrose and related sugar solution, and glycols such as, propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

When the inhibitor is deferoxamine, deferoxamine compositions forparental use can be in the form of a solution or a suspension. Suchsolutions or suspenions may also include sterile diluents such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents. Parenteralformulations may also include antibacterial agents such as benzylalcohol or methyl parabens, or antioxidants such a sodium bisulfite.Buffers such as acetates, citrates, or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose may also beadded. The parenteral preparation can be enclosed in ampules, disposablesyringes, or multiple dose vials made of glass or plastic.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

Slow-release deferoxamine compositions for intramuscular administrationmay be formulated by standard methods, such as a microcrystallinecomposition. Deferoxamine preparations with longer half-lives may beformulated by conjugation of deferoxamine with, for example, dextrans orpolyethylene glycols. In addition, deferoxamine derivatives with greatability to permeate cell membranes can be made by linking deferoxamineto a lipophilic ester moiety such as acetyoxymethyl ester, which is thenremoved by intracellular esterases once the compound is inside the cell(Meijler et al., “Synthesis and Evaluation of Iron Chelators with MaskedHydrophilic Moieties”, J. Am. Chem. Soc. 124:12666-12667 (2002)).

The formulations containing the inhibitors of the present invention mayalso be administered directly to the airways in the form of an aerosol.For use as aerosols, the inhibitors of the present invention in solutionor suspension may be packaged in a pressurized aerosol containertogether with suitable propellants, for example, hydrocarbon propellantslike propane, butane, or isobutane with conventional adjuvants. Theinhibitors of the present invention also may be administered in anon-pressurized form such as in a nebulizer or atomizer.

In carrying out this method, an ROS-mediated injury can be treated orprevented. Hyperglycemic conditions which can be so treated or preventedinclude chronic hyperglycemia. This includes hyperglycemic diabetes oracute hyperglycemia (such as stress hyperglycemia). Resistance toinsulin is another form of a metabolite-induced excessive ROS productionin accordance with this aspect of the present invention. This can bewhere there is resistance to insulin resulting in increased free fattyacid flux and increased free fatty acid oxidation by vascular cells.

Another aspect of the present invention relates to a method of promotingneovascularization in a subject prone to hyperglycemia or increasedfatty acid flux. This method involves administering an ROS inhibitor tothe subject under conditions effective to promote neovascularization inthe subject.

Here, neovascularization can be in response to hypoxic signaling, andinvolve both angiogenesis (e.g. cardiac or lower limb) orvasculogenesis. The subject can have an ischemic condition, such ascoronary artery disease, peripheral vascular disease, cerebral vasculardisease, or a wound which is either chronic or acute.

The ROS inhibitor, its formulation, and its modes of administration forthis embodiment of the present invention are the same as those describedabove.

Here the subject is preferably a human prone to hyperglycemia or fattyacid flux.

A further aspect of the present invention pertains to a methodinhibiting oxidation or excessive release of free fatty acids in asubject. This method involves administering to the subject certaincompounds under conditions effective to inhibit oxidation excessiverelease of free fatty acids in the subject. These compounds includethiazolidinedione, nicotinic acid, etomoxir, and ranolazine.

In this embodiment of the present invention, the above-identifiedcompounds are formulated and administered in substantially the same wayas noted above.

In this aspect of the present invention, the subject is a mammal,preferably a human.

A further aspect of the present invention is directed to a method ofidentifying compounds suitable for treatment or prevention ofROS-mediated injury. This method involves providing a diabetic animalmodel and inducing diabetes in the animal model. A compound to be testedis then administered to the animal model. Compounds which achieverecovery of local oxygen tension, blood flow increase in vessel density,and tissue survival in the animal model as therapeutic candidates fortreating or preventing ROS-mediated injury are then recovered.

EXAMPLES Example 1—Three Different Murine Models of Diabetes ExhibitIncreased Tissue Necrosis in Response to Ischemia

It is well recognized that diabetic tissues have a reduced tolerance toischemia (Haffner et al., “Mortality From Coronary Heart Disease inSubjects With Type 2 Diabetes and in Nondiabetic Subjects With andWithout Prior Myocardial Infarction,” N Engl J Med 339:229-34 (1998);Jude et al., “Peripheral Arterial Disease in Diabetic and NondiabeticPatients: a Comparison of Severity and Outcome,” Diabetes Care 24:1433-7(2001); Tuomilehto et al., “Diabetes Mellitus as a Risk Factor for DeathFrom Stroke. Prospective Study of the Middle-Aged Finnish Population,”Stroke 27:210-5 (1996); Waltenberger, “Impaired Collateral VesselDevelopment in Diabetes: Potential Cellular Mechanisms and TherapeuticImplications,” Cardiovasc Res 49:554-60 (2001); Rivard at al., “Rescueof Diabetes-Related Impairment of Angiogenesis By Intramuscular GeneTherapy With Adeno-VEGF,” Am J Pathol 154:355-63 (1999); Kip at al.,“Differential Influence of Diabetes Mellitus on Increased JeopardizedMyocardium After initial Angioplasty or Bypass Surgery: BypassAngioplasty Revascularization Investigation,” Circulation 105:1914-20(2002); Partamian at al., “Acute Myocardial Infarction in 258 Cases ofDiabetes. Immediate Mortality and Five-Year Survival,” N Engl J Med273:455-61 (1965); Simovic et al., “Improvement in Chronic IschemicNeuropathy After Intramuscular phVEGF165 Gene Transfer in Patients WithCritical Limb Ischemia,” Arch Neurol 58:761-8 (2001): Margolis at al.,“Risk Factors for Delayed Healing of Neuropathic Diabetic Foot Ulcers: APooled Analysis,” Arch Dermatol 136:1531-5 (2000), which are herebyincorporated by reference in their entirety). Clinically, this resultsin increased rates of heart failure, increased mortality and prolongedwound healing. While this relationship has been studied in animal modelsof cardiac and hindlimb ischemia (Rivard at al., “Rescue ofDiabetes-Related Impairment of Angiogenesis By Intramuscular GeneTherapy With Adeno-VEGF,” Am J Pathol 154:355-63 (1999); Schratzberger,et al., “Reversal of Experimental Diabetic Neuropathy by VEGF GeneTransfer,” J Clin Invest 107:1083-92 (2001), which are herebyincorporated by reference in their entirety), there are limitations tothese models. Due to the variations in large vessel anatomy, theresultant pattern of necrosis is unpredictable, leading to discrepanciesin the experimental results. In addition, it is not possible todetermine tissue survival except at sacrifice. Furthermore, indirectmeasures of perfusion such a laser doppler must often be utilized toestimate ischemia, but these techniques do not provide directinformation regarding tissue oxygenation.

To address these problems, a novel model of graded ischemia in thedorsal soft tissue of mice has been created (FIG. 5) (Tepper et al.,“Human Endothelial Progenitor Cells From Type II Diabetics ExhibitImpaired Proliferation, Adhesion, and incorporation Into VascularStructures,” Circulation 106:2781-6 (2002), which is hereby incorporatedby reference in its entirety). Since the vascular anatomy of the mousedorsum is precisely known, and the major axial vessels can be easilyvisualized, one can create a reliable zone of ischemia with areproducible oxygen gradient in the tissue. This has been confirmed withdirect tissue oxygen tension measurements utilizing five referencepoints (p1-p5) spaced 0.5 cm apart proceeding from the least to mostischemic regions. This also allows for the study of discretemicroenvironments of ischemia (Areas A, B, C), with Area A being theleast ischemic and Area C being the most ischemic portion of the softtissue. The design of this model facilitates direct dynamic measurementof oxygen tension, quantitation of tissue survival, with a degree ofreproducibility that allows correlation of specific oxygen tensions withchanges in gene expression.

Using this model, it has been observed that the response too ischemia isdramatically impaired in three different murine models of diabetes, allcharacterized by significant hyperglycemia. In the db/db mouse, a leptinreceptor deficient model of Type U diabetes, it has been demonstratedthat ischemia produces significant necrosis of nearly all of the tissue,whereas all the tissue survived in non-diabetic animals. Similar resultswere noted in the streptozotocin-induced diabetic mouse model (Stz), aswell as an Akita mouse model of Type I diabetes with tissue survivalapproximately 30% of that observed in non-diabetic mice (FIG. 6).Importantly, oxygen tensions and vascular density (as determined by CD31staining and FITClectin perfusion) were identical in all four groupsprior to surgery, suggesting that the differences in tissue survivalwere due to an impaired response to ischemia rather than baselinedifferences in vascular density.

Example 2—Diabetic Mice have a Diminished Neovascular Response toIschemia

The decrease in tissue survival observed in this model was alsoassociated with diminished neo-vascularization in the surviving tissue.Seven days following surgery, the oxygen tension in ischemic soft tissueof non-diabetic mice approaches that of normal skin (FIG. 7, grey plot),while the diabetic mice demonstrate a significant reduction in oxygentension at the same reference points (black plot). These findingscorrelated with a reduction in the number of blood vessels observed inthe surviving tissue in diabetic mice (FIG. 8, black plot) as determinedby CD31 staining. This suggests that ischemia-induced neovascularizationis impaired in diabetic mice.

Example 3—Prevention of Hyperglycemia-Induced Reactive Oxygen SpeciesRestores Tissue Survival in a Diabetic Animal Model

It has been examined whether increased oxidative damage was an upstreammodulator of the impaired tissue response to ischemia in diabeticanimals. To address this question, a transgenic mouse that overexpressmitochondrial manganese superoxide dismutase (MnSOD) was used. MnSODcatalyzes the formation of molecular oxygen from superoxide, preventingthe generation of ROS, and effectively blocks all four pathways ofhyperglycemic damage. Diabetes was induced in wild type and MnSODtransgenic mice via streptozotocin injection, and hyperglycemia (>400mg/dl) was maintained for one month. Following ischemic surgery, tissuewas monitored by direct oxygen tension measurements on days 1, 3, and 7.Compared to wild type diabetic mice, MnSOD diabetic mice demonstrated arapid recovery of local tissue oxygen tensions, neovascularization, andincreased tissue survival that was similar to that observed innon-diabetic mice (FIG. 9). Non-diabetic MnSOD control mice were similarto wild type mice. This suggests that the prevention ofhyperglycemia-Induced ROS improves tissue survival in diabetic animalsfollowing ischemic events.

Example 4—Chronic High Glucose Levels Also Correlate with IncreasedMitochondrial Membrane Potential

The effects of high glucose culture on mitochondrial membrane potentialwere also examined in C2CI 2 cells exposed to acute or chronic highglucose using the potential-dependent cationic dye JC-1. This has beenused as an indicator of oxidative stress. In concordance with recentreports (Du et al., “Hyperglycemia Inhibits Endothelial Nitric OxideSynthase Activity by Posttranslational Modification at the Akt Site,” JClin Invest 108:1341-8 (2001), which is hereby incorporated by referencein its entirety), chronic high glucose profoundly increases themitochondrial proton electrochemical gradient (evidenced by a shift toorange-red fluorescence) compared to normal glucose culture or acuteexposure to high glucose (FIG. 10) (Du et al., “Hyperglycemia InhibitsEndothelial Nitric Oxide Synthase Activity by PosttranslationalModification at the Akt Site,” J Clin Invest 108:1341-8 (2001), which ishereby incorporated by reference in its entirety). Thus, a correlationexists between hyperglycemia, oxidative stress, and VEGF impairment invitro.

Example 5—Impaired VEGF Production Lies at the Level of RNATranscription

With evidence implicating decreased VEGF production as a contributor toimpaired angiogenesis in hyperglycemic states, the mechanism by whichhigh glucose alters VEGF expression was examined. Analysis of VEGF mRNAtranscripts present in normal and high glucose culture under hypoxicconditions revealed a substantial reduction in VEGF mRNA production incells cultivated in high glucose (FIG. 11). Possible explanations forthis finding included abnormal mRNA stabilization or decreased promoteractivity in high glucose. To address the issue of mRNA stabilization,the RNA ½-life in C2Cl2 myoblasts was examined by inhibitingtranscription with actinomycin D. Results of these experiments showed nodifferences in VEGF mRNA stability between normal an hyperglycemic cellsdespite significant differences in VEGF protein levels (FIG. 12). VEGFpromoter activity was then examined using a reporter constructcontaining the full length VEGF promoter fused to a luciferase gene.This construct was transiently co-transfected into C2Cl2 myoblastscultivated in normal and high glucose with a constitutively expressedRenilla plasmid to control for transfection efficiency. Hypoxia-inducedluciferase production was significantly impaired in high glucoseconditions compared to normal glucose controls (FIG. 13). Thisdemonstrates that the impaired VEGF protein production in hypoxiaresulted from decreased VEGF transcription in vivo.

Example 6—p300 and HIF-1a are Substrates for 0-linked Glycosylation,Potentially Linking the Hexosamine Pathway of Hyperglycemic OxidativeDamage to Impairments in Hypoxia-Induced VEGF Expression

Based on findings implicating impaired HIF-1α transactivation in highglucose as the mechanism for impaired hypoxia-induced VEGF expression,potential post-translational modifications of HIF-1α were examined underthese conditions. It was initially examined whether HIF-1α is asubstrate for O-linked glycosylation. HIF-1α was immunoprecipitated fromcells grown in normal or high glucose conditions, and Western blots wereprobed with an antibody that specifically recognizes residues containingthe O-linked glycosylation modification. While no glycosylated HIF-1αwas present under normal glucose conditions, there was significantglycosylation in high glucose (FIG. 14). This is the first demonstrationthat HIF-1α is a substrate for O-linked glycosylation, and ispreferentially glycosylated under conditions of high glucose.

Since the HIF-1 transcriptional complex is comprised of severalcoactivators, it was also examined whether p300, the major co-activatorof the HIF-1, was also glycosylated. While many transcription factorshave been found to associate with p300 constitutively, some cases havebeen identified where this interaction is modulated bypost-translational modification (Zanger at al., “CREB Binding ProteinRecruitment to the Transcription Complex Requires GrowthFactor-Dependent Phosphorylation of its OF Box,” Mol Cell 7:551-8(2001); Soutoglou at al., “Acetylation Regulates Transcription FactorActivity at Multiple Levels,” Mol Cell 5:745-51 (2000), which are herebyincorporated by reference in their entirety). Repeating the HIF-1experiments, it was also found that p300 also serves as a substrate forpost-translational O-linked glycosylation in conditions of high glucose.This was physiologically significant since the association of p300 withthe transcription factor peroxisome proliferator-activated receptorgamma (PPARγ) was reduced in conditions of high glucose compared tonormal glucose by co-immunoprecipitation assays (FIG. 15A-B).Interestingly, blockade of the rate-limiting enzyme of hexosaminebiosynthesis, glutamine:fructose-6-phosphate amidotransferase (GFAT)with antisense oligonucleotides reduced the amount of Olinkedglycosylation of p300 in high glucose nearly three-fold, and restoredthe p300/PPARγ interaction to levels comparable to cells grown in normalglucose (FIG. 15A-B). This suggests that the recruitment of p300 totranscriptional complexes is impaired in conditions of high glucose,which can be reversed by preventing glucose-induced O-linkedglycosylation. The physiologic relevance of O-linked glycosylation ofHIF-1α is unclear. However, the demonstration that glycosylationmodifies p300 function suggests a possible mechanism by which the HIF-1transcriptional complex fails to upregulate VEGF expression, due to itsinability to recruit and/or associate with co-activators required fortranscriptional activation (i.e. p300).

Example 7—Hyperglycemia-Induced Reactive Oxygen Species ActivatePathways of Cellular Damage, Impairing Endothelial Cell Function

The data presented thus far have examined the mechanisms responsible forinitial observations that high glucose levels, both in vivo and invitro, produce profound deficits in the ability to upregulate VEGF underhypoxic conditions.

Although there is significant literature examining hyperglycemia-inducedvascular damage in non-ischemic settings, very few studies have examinedthe effect of hyperglycemia-induced cellular damage on vascularfunctions in ischemic settings. This is of clinical importance, as mostsituations requiring new vascular growth occur in scenarioscharacterized by significant tissue hypoxia. It has been demonstratedthat endothelial cells grown in high glucose in vitro show increasedmitochondrial production of ROS. This results in increased hexosaminepathway activity with increased glycosylation of certain transcriptionfactors (SPI) and signaling molecules (eNOS), increased PKC activityresulting in part in increased NFkB activity, greater accumulation ofAGEs, and increased flux through the sorbitol pathway (FIG. 16A-C). Thedownstream consequences of these intracellular events likely result inimpaired neovascularization observed in vivo, but the intermediate stepsremain unclear.

Example 8—Diabetic Cells are Impaired in Functions Critical forAngiogenesis

While it is clear that VEGF expression is altered in diabetic states, ithas also been demonstrated that diabetic cells are impaired in otherways. Fibroblasts isolated from diabetic mice (db/db) show dramaticdecreases in migration (four-fold less) than normal fibroblasts oncollagen and fibronectin using a gold salt phagokinetic migration assay.When the haplotactic response of these cells was examined using amodified Boyden chamber migration assay, a similar decrease of 77% inmigration in response to serum and PDGF was observed (Lerman et al.,“Cellular Dysfunction in the Diabetic Fibroblast: Impairment inMigration, Vascular Endothelial Growth Factor Production, and Responseto Hypoxia,” Am J Pathol 162-303-12 (2003), which is hereby incorporatedby reference in its entirety).

Once again, this difference was accentuated by hypoxia (FIG. 17A-B).Whereas migration in normal cells was upregulated by hypoxia (two-fold),diabetic cells showed no difference in the rate of migration in hypoxia.These assays again emphasize the profound impact that diabetes has oncellular function, and that this impact is magnified under hypoxicconditions.

These migration differences may be due to differential expression ofmembers of the matrix metalloproteinase (MMP) family in diabeticfibroblasts. It has been demonstrated that diabetic fibroblasts havegreater levels of pro-MW-9 than normal fibroblasts, but no differencesin active MMP-9 or active/pro-MMP-2. (FIG. 18A-B). This confirms similarfindings in endothelial cells cultured in high glucose (Uemura at al.,“Diabetes Mellitus Enhances Vascular Matrix Metalloproteinase Activity:Role of Oxidative Stress,” Circ Res 88:1291-8 (2001), which is herebyincorporated by reference in its entirety). Furthermore, these findingssuggest that diabetic cellular dysfunction is not characterized by asimple downregulation of all cellular proteins or functions, butinvolves selective modulation of specific genes and proteins.

Example 9—Endothelial Progenitor Cells from Type II Diabetic Patientsare Impaired in their Ability to Proliferate, Adhere, and Incorporateinto Vascular Structures

Hyperglycemic alterations in the effector cells of vasculogenesis, theendothelial progenitor cell or precursor cell remain poorly defined.Recently, it was demonstrated that endothelial progenitor cells (EPCs)harvested from Type 1 diabetic patients exhibit reduced proliferation,adhesion, and incorporation into vascular structures as compared to agematched controls under normoxic conditions (Tepper et al., “HumanEndothelial Progenitor Cells From Type II Diabetics Exhibit ImpairedProliferation. Adhesion, and incorporation Into Vascular Structures,”Circulation 106:2781-6 (2002), which is hereby incorporated by referencein its entirety). Diabetic cultures contained significantly fewer EPCsafter 7 days of expansion (FIG. 19A-D), and this was inverselycorrelated with HbA_(1c). Additionally, significantly fewer EPC-bearingclusters were noted in the cultures of diabetic patients. This wasinversely correlated with the number of years of clinical diabetes(R=−0.471, P<0.01). Functionally, these cells were found to adhere lessto TNF-α activated endothelial monolayers but exhibited normal adhesionto quiescent endothelial monolayers, which suggests that their abilityto respond to environmental cues is deficient. This was confirmed within vitro angiogenesis assays, which demonstrated that fewer diabeticEPCs were incorporated into tubules on Matrigel when compared toage-matched controls.

Example 10—Endothelial Progenitor Cells from Diabetic Patients have anImpaired Ability to Respond to Hypoxia

Given preliminary data suggesting that diabetic cells have an impairedresponse to hypoxia, studies in EPCs have been to specifically examinethe response of these cells to an ischemic environment. It wasdemonstrated that EPCs from Type II diabetic patients were impaired intheir ability to adhere to hypoxic endothelial monolayers, migratetowards conditioned media from hypoxic endothelial cells, andproliferate a hypoxic environment (FIG. 10A, B, C, respectively). Thismay be reflective of an impaired ability of these cells to sense andrespond appropriately to hypoxic environmental cues, resulting in poorneovascularization.

Example 11—Deferoxamine Prevents Hyperglycemia-Induced Reactive OxygenProduction in Vascular Endothelial Cells

Cultured vascular endothelial cells were treated with deferoxamine todetermine the effect of deferoxamine on hyperglycemia-induced reactiveoxygen production by those cells.

Cell culture conditions: For ROS measurement, bovine aortic endothelialcells (BAECs, passage 4-10) were plated in 96 well plates at 100,000cells/well in Eagle's MEM containing 10% FBS, essential and nonessentialamino acids, and antibiotics. Cells were incubated with either 5 mMglucose, 30 mM glucose, 30 mM glucose plus 100 micromolar deferoxamine,30 mM glucose plus 250 micromolar deferoxamine. The deferoxamine wasfreshly prepared and added to the cells on three consecutive days. TheROS measurements were performed 72 hrs after the initial treatment.

Intracellular reactive oxygen species measurements: The intracellularformation of reactive oxygen species was detected using the fluorescentprobe CMH2DCFDA (Molecular Probes). Cells (1×105 ml-l) were loaded with10 μM CM-H2DCFDDA, incubated for 45 rain at 37° C., and analyzed in anHTS 7000 Bio Assay Fluorescent Plate Reader (Perkin Elmer) using theHTSoft program. ROS production was determined from an H202 standardcurve (10-200 nmol ml-l).

As shown in FIG. 21 deferoxamine inhibited production of ROS in vascularendothelial cells in culture. Diabetic levels of hyperglycemia causeincreased ROS (superoxide) production in these cells (FIG. 21, bar 2).Adding 250 μM deferoxamine completely prevents this damaging effect(FIG. 21, bar 4).

Thus, the iron chelator deferoxamine has a profound effect on vascularendothelial cells—i.e. it prevents completely hyperglycemia-inducedoverproduction of hydroxyl radicals (FIG. 21).

Example 12—Normalizing Excess Mitochondrial Superoxide ProductionInhibits Hyperglycemia-Induced Increases in Intracelluar Free Iron inAortic Endothelial Cells

For free intracellular iron measurement, bovine aortic endothelial cells(“BAECs”, passage 4-10) were plated in 24 well plates at 500,000cells/well in Eagle's MEM containing 10% FBS, essential and nonessentialamino acids, and antibiotics. Cells were infected with UCP-1, Mn-SOD orempty adenoviral vectors, respectively, for 48 hours. 30 mM glucose wasadded to each well that was infected with the adenovirus Uninfectedcells were incubated with 5 mM and 30 mM glucose as controls. The freeintracellular iron was detected after 24 hours.

In order to detect intracellular free iron, cells were loaded withfura-2 AM in the dark at 37° C. for 15 min in 1 ml of TBSS containing 5μM furs-2 AM. After loading, cells were incubated with TBSS with 1 ml of20 μM EDTA for 5 min. (Kress et al., “The Relationship betweenIntracellular Free Iron and Cell Injury in Cultured Neurons, Astrocytes,and Oligodendrocytes”, J. Neuro., 22(14):5848-5855 (2002), which ishereby incorporated by reference in its entirety). Fluorescence wasdetected using an Olympus IX70 with 10× planapo objectives, run by I.P.Lab Spectrum on a Power PC computer. Analysis was performed with I.P.Lab Spectrum.

As shown in FIG. 22, bar 2, hyperglycemia, increased the amount of freeiron by nearly 3-fold. Since the probe fura-2 AM specifically detectsFe³⁺ iron, this shows that it is free Fe³⁺ iron which is increased.Inhibition of this effect by overexpression of uncoupling protein-1, amitochondrial protein that prevents superoxide formation by the electrontransport chain (bar 3) demonstrates that the mitochondria are theorigin of the hyperglycemia-induced-superoxide. Inhibition of thiseffect by overexpression of MnSOD, the mitochondrial isoform of theenzyme superoxide dismutase (bar 4), demonstrates that mitochondrialsuperoxide is the reactive oxygen species that induces increasedintracellular free iron.

Example 13—Deferoxamine Inhibits Hyperglycemia-Induced Increases inIntracellular Free Iron in Aortic Endothelial Cells

Bovine aortic endothelial cells (“BAECs”, passage 4-10) were plated in24 well plates at 500,000 cells/well in Eagle's MEM containing 10% FBS,essential and nonessential amino acids, and antibiotics. Cells wereincubated with either 5 mM glucose, 30 mM glucose, or 30 mM glucose plus100 μM deferoxamine. Free intracellular iron measurement was performed24 hours later. To detect intracellular free iron, cells were loadedwith fura-2 AM as described above in Example 12.

As shown in FIG. 23B, hyperglycemia (accomplished by 30 mM glucoseincubation) dramatically increases intracellular free iron in the Fe³⁺form compared to normal glycemia, as shown in FIG. 23A (accomplished by5 mM glucose treatment), as it did in Example 12. As shown in FIG. 23C,the Fe³⁺-specific iron chelator deferoxamine (100 μM) completelyprevents this effect of hyperglycemia.

Example 14—Deferoxamine and the Hydroxyl Radical Scavenger DMSO BothInhibit Hyperglycemia-Induced Increases in DNA Strand Breakage in AorticEndothelial Cells

Bovine aortic endothelial cells (“BAECs”, passage 4-10) were plated in10 mm cell culture plates until confluent. Cells were incubated witheither 5 mM glucose, 30 mM glucose, 30 mM glucose plus 100 μMdeferoxamine (DFO), or 30 mM glucose plus 100 μM DMSO, a hydroxylradical scavenger, for 7 days. Medium with reagents was changed daily.DNA strand breakage was detected using the Comet assay method.

DNA breakage detection was performed using the Comet Assay kit (TrevigenGaithersburg Md.). Briefly, single cell electrophoresis was performed onthe cometslide for 10 min at 1 volt/cm (measured from one electrode toanother). After air-drying, the cometslide was stained with SYBR green.Fluorescence was detected using the Olympus IX70 fluorescent microscopeand analysis of the fluorescent density of DNA breakage (length of tail)was performed using Image J software.

It has previously been shown that hyperglycemia-Induced superoxideproduction by the mitochondrial electron transport chain causes DNAstrand breakage in aortic endothelial cells, as demonstrated in FIG.24B. The data shown in FIG. 24C prove that this effect requires thesuperoxide-induced increase in free Fe 3⁺. Similarly, the data shown inFIG. 24D show that this effect requires superoxideinduced hydroxylradical production. Together, these data show that deferoxaminetreatment prevents hydroxyl radical generation and subsequent DNA strandbreakage, despite the continued overproduction of superoxide by themitochondrial electron transport chain.

Example 15—Deferoxamine and the Hydroxyl Radical Scavenger DMSO BothInhibit Hyperglycemia-Induced Increases in PARP Activity in AorticEndothelial Cells

Bovine aortic endothelial cells (“BAECs”, passage 4-10) were plated in10 mm cell culture plates until confluent. Cells were incubated witheither 5 mM glucose, 30 mM glucose, 30 mM glucose plus 100 μMdeferoxamine, or 30 mM glucose plus 100 μM DMSO for 6 days and mediumchanged daily.

The 3H-NAD incorporation method was used to assess PARP activity. BAECswere incubated with buffer which was composed of 56 mM Hepes (pH 7.5),28 mM KCl, 28 mM NaCl, 2 mM MgCl₂, 0.01% digitonin, 25 mM NAD⁺, and 1μCi/ml ³HNAD⁺ for 10 min at 37° C. TCA was added to precipitateribosylated protein and cells were lysed in 2% NaOH. Detection ofincorporated ³H-NAD was performed using a scintillation counter, andPARP activity determined according to the number of ³H-NAD dpm.

It has previously been shown that hyperglycemia-induced superoxideproduction by the mitochondrial electron transport chain causes DNAstrand breakage which then activates the enzyme poly(ADP-ribose)polymerase (PARP) in aortic endothelial cells, as shown inFIG. 25. The data shown in bar 3 prove that this effect requires thesuperoxide-induced increase in free Fe³⁺. Similarly, the data shown inbar 4 show that this effect requires superoxide-induced hydroxyl radicalproduction. Together, these data show that deferoxamine treatmentprevents hydroxyl radical generation, subsequent DNA strand breakage,and resultant PARP activation, despite the continued overproduction ofsuperoxide by the mitochondrial electron transport chain.

Example 6—Deferoxamine Prevents Hyperglycemia-Induced Inhibition ofProstacyclin Synthase (PGF-1a) in Aortic Endothelial Cells

Bovine aortic endothelial cells (“BAECs”, passage 4-10) were plated in a24-well plate (50,000 cell/well). Cells were incubated with either 5 mMglucose, 30 mM glucose, or 30 mM glucose plus 100 μM deferoxamine. Theprostacyclin synthase product, PGF-1α, was measured 24 hours later.

Prostacyclin synthase activity measured as the concentration of thestable product of prostacyclin synthase, PGF-1a. A competitiveimmunoassay method (Correlate-EIA) was used for the quantitativedetermination of 6-keto-PGFIα. Samples (100 μl) collected from BAECsculture medium were added to the assay plate, which was precoated withantibody (6-keto-PGF1α, EIA conjugate solution). PGFIα concentration wascalculated according to a standard curve, and data analysis performedusing AssayZap software.

It has previously been shown that hyperglycemia-induced superoxideproduction by the mitochondrial electron transport chain completelyinactivates the endothelial enzyme prostacyclin synthase, which is amajor natural defense against the development of atherosclerosis. In bar2 of FIG. 26, hyperglycemia is shown to decrease the activity of thisenzyme by over 90%. In contrast, bar 3 shows that hyperglycemia does notinhibit the activity of this important antiatherogenic enzyme at allwhen the superoxide-induced increase in free Fe³⁺ is prevented bydeferoxamine.

Example 17—Deferoxamine Prevents Diabetes-Induced Inhibition ofProstacyclin Synthase (PGF-1a) In Aortas of Diabetic Mice

Male C57B16 mice (6-8 weeks old) were made diabetic by daily injectionsof S0 mg/kg streptozotocin in 0.05 M NaCitrate pH 4.5 after an eighthour fast, for five consecutive days. Two weeks after the initialinjection the blood glucose was determined and the diabetic mice wererandomized into two groups with equal mean blood glucose levels.Deferoxamine (10 mg/kg) was injected subcutaneously once per day for 7days in one group of diabetic animas. The aortas were collected, forprostacyclin synthase activity measurement.

Prostacyclin synthase activity measurement A competitive immunoassaymethod (Correlate-EIA) was used for the quantitative determination of6-keto-PGF_(1α). Mouse aortas were washed with PBS and incubated at 37°C. for 3 hours in 400 μl incubation buffer containing 20 mM TRIS buffer(pH 7.5), and 15 μM arachidonic acid. 100 μl of sample was used tomeasure the PGF1α.

It has previously been shown that diabetes-induced superoxide productionby the mitochondrial electron transport chain completely inactivates theendothelial enzyme prostacyclin synthase in aortas of diabetic mice. Inbar 2 of FIG. 27, hyperglycemia is shown to decrease the activity ofthis enzyme in vivo by over 90%. In contrast, bar 3 of FIG. 27 showsthat hyperglycemia does not inhibit the activity of this importantantiatherogenic enzyme at all when the superoxide-induced increase infree Fe³⁺ is prevented by deferoxamine.

Example 18—Deferoxamine Prevents Hyperglycemia-Induced Inhibition orEndothelial Nitric Oxide Synthase (eNOS) in Aortic Endothelial Cells

Bovine aortic endothelial cells (“BAECs”, passage 4-10) were plated in24-well plate (50,000 cell/well). Cells were incubated with either 5 mMglucose, 30 mM glucose alone, or 30 mM glucose plus 100 μM deferoxamine,for 24 hour. Six hours before eNOS activity determination, media withoutarginine was added to the cells to deplete endogenous arginine.

Measurement of eNOS activity was accomplished as follows. BAECs wereincubated with 400 μl of PBS-³H-arginine (1.5 μci/ml) buffer for 30 minat 37° C. The reaction was stopped by adding IN TCA (500 μl/well, icecold), the cells were freeze fractured in liquid nitrogen for 2 min andthawed at 37° C. for 5 min to obtain the cell lysate. After extractionwith ether, the cell lysate was adjusted to pH 5.5 using Hepes buffercontaining 2 mM EDTA and 2 mM of EGTA, then loaded onto Trisformed DOWEX50WX8 ion-exchange columns and ³H-citrulline collected. Detection of³H-citrulline was performed using a liquid scintillation counter, andeNOS activity was calculated from the amount of ³H-citrulline generated.

It has previously been shown that hyperglycemia-induced superoxideformation significantly inactivates another critical endothelial enzyme,endothelial nitric oxide synthase (eNOS). This enzyme plays a criticalrole in acute dilation of blood vessels in response to hypoxia, and achronic role as another major defense against development andprogression of atherosclerosis. In bar 2 of FIG. 28, hyperglycemia isshown to decrease eNOS activity by 65%. In contrast, bar 3 shows thathyperglycemia does not inhibit the activity of this importantantiatherogenic enzyme at all when the superoxide-induced increase infree Fe 3⁺ is prevented by deferoxamine.

Example 19—Deferoxamine Prevents Diabetes-Induced Inhibition ofEndothelial Nitric Oxide Synthase (eNOS) in Aortas of Diabetic Mice

Male C57B16 mice (6-8 weeks old) were made diabetic by daily injectionsof 50 mg/kg streptozotocin in 0.05 M NaCitrate pH 4.5 after an eighthour fast, for five consecutive days. Two weeks after the initialinjection, the blood glucose was determined and the diabetic mice wererandomized into two groups with equal mean blood glucose levels.Deferoxamine (10 mg/kg) was injected subcutaneously once per day for 7days in one group of diabetic animals. The aortas were collected forendothelial nitric oxide synthase (eNOS) activity measurement.

Measurement eNOS activity was accomplished as follows. Aortas werecollected in liquid-nitrogen and tissue proteins isolated.Immunoprecipitation methods were used to purify the eNOS from tissuelysates. The purified eNOS immuno-complex was incubated with 100 μl ofreaction buffer (3 μM Tetrahydrobiopterin, 1 mM NAPDH, 2.5 mM CaCl₂, 200U Calmodulin, ³H-L-arginine 0.2 μCi) for 45 min at 37° C. with rolling.After the incubation, samples were loaded onto Tris-formed DOWEX 50WX8ion-exchange column and ³H-citrulline was collected. ³H-citrulline wasquantitated using a liquid scintillation counter and eNOS activity wascalculated from the amount of ³H-citrulline generated.

It has previously been shown that diabetes-induced-induced superoxideproduction by the mitochondrial electron transport chain inactivates theendothelial enzyme eNOS in aortas of diabetic mice. In bar 2 of FIG. 29,diabetic hyperglycemia is shown to decrease the activity of this enzymein vivo by 65%. In contrast, bar 3 shows that hyperglycemia does notinhibit the activity of this important antiatherogenic enzyme at allwhen the superoxide-induced increase in free Fe³⁺ is prevented bydeferoxamine.

Example 20—Diabetes-Induced Defect in Angiogenic Response to Ischemia

FIGS. 30A-B show that diabetic animals do not increase oxygenation byforming new vessels the way non-diabetic animals do. FIGS. 30C-D showthat diabetic animals only mobilize 0.22 vs. 1.83% of bonemarrow-derived endothelial precursor cells in response to ischemia.FIGS. 30E-F show (black bar) that diabetics do not increase capillaryformation in ischemic tissue.

Researchers have created a novel model of graded ischemia in the dorsalsoft tissue of mice. Since the vascular anatomy of the mouse dorsum isprecisely known, and the major axial vessels can be easily visualized,this model creates a reliable zone of ischemia with a reproducibleoxygen gradient in the tissue. This has been confirmed with directtissue oxygen tension measurements utilizing four reference points(p1-p4) spaced 0.5 cm apart, proceeding from the least to most ischemicregions.

The mechanisms underlying this diabetes-induced defect are complex andincompletely understood, but appear to involve mitochondrial superoxideoverproduction, since the defect is significantly prevented in diabetictransgenic mice which overexpress the mitochondrial isoform of SOD.

Example 21—Deferoxamine Treatment Corrects the Diabetes-Induced Defectin Angiogenic Response to Ischemia

The effect of deferoxamine, an iron chelator, on ischemicneovascularization in streptozotocin-induced diabetic (STZ) and wildtype C57 (WT) mice was examined. Male C57B16 mice (6-8 weeks old) weremade diabetic by daily injections of 50 mg/kg streptozotocin in 0.05 MNaCitrate pH 4.5 after an eight hour fast, for five consecutive days.Two weeks after the initial injection the blood glucose was determined,the diabetic mice were randomized into two groups with equal mean bloodglucose levels.

The treatment group was pretreated 7 days prior to having an ischemicflap created on their dorsum and throughout the experiment with dailyinjections of deferoxamine (10 mg/kg) subcutaneously once per day for 7days in one group of diabetic animals.

On day 7, it was found that blood flow was restored to normal in theSTZ-deferoxamine group (DM+DEF) when compared with the non-diabeticuntreated group (WT), end the severely impaired STZ-untreated group(DM), a assessed by Doppler and as shown in FIG. 31A. FIG. 31B showstissue survival was restored to normal in the STZ-deferoxamine group(DM+DEF) when compared with the non-diabetic untreated group (WT), andthe severely impaired STZ-untreated group (DM). CD31 positive bloodvessel counts demonstrate that post-ischemic neovascularization wasrestored in the STZ-deferoxamine group (STZ DefC), as shown in FIG. 32.Interestingly, deferoxamine in the wild type mice (WT Def C) alsoimproved neovascularization. EPC mobilization was also improved in theSTZ-deferoxamine group when compared with the untreated STZ mice.Migration of diabetic bone marrow derived, lineage depleted cellpopulation migration toward SDF was restored to normal when STZ micewere treated with deferoxamine. See FIG. 35.

These results show that treatment of diabetic animals with deferoxaminecompletely prevents the diabetes-induced defect in the normal angiogenicresponse to ischemia.

Example 22—Deferoxamine Normalizes Diabetic Wound Healing andDiabetes-Induced Defect in Angiogenic Response to Ischemia

The effect of reducing the wound healing in diabetic mice (db/db) bytreating them with deferoxamine was also studied in the novel model ofgraded ischemia in the dorsal soft tissue of mice of Example 20. Theanimals were made diabetics as described in Example 21.Deferoxamine-treated diabetic mice demonstrated complete would closureat day 16, whereas untreated db mice did not close their wounds untilday 26 (FIGS. 33A-B). FIGS. 34 A-B show that diabetic animals do notincrease oxygenation by forming new vessels the way deferoxamine-treateddiabetic mice do. FIGS. 34C-D show that deferoxamine-treated diabeticanimals mobilize 1.12% of bone marrow-derived endothelial precursorcells in response to ischemia compared to 0.22% of diabeticdeferoxamine-untreated mice. FIG. 348-F show (black bar) thatdeferoxamine-treated diabetics mice substantially increase capillaryformation in ischemic tissue compared to diabetic deferoxamine-untreatedmice

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions and the like can bemade without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1-19. (canceled)
 20. A composition of matter comprising the compoundR-L-C-M or a pharmaceutically acceptable salt thereof, wherein R is H ora biocompatible moiety capable of facilitating or hindering thepenetration of said composition of matter in target cells, L is a linkerconnecting R to C, wherein L is capable of facilitating a rapid cellularintake and delaying a cellular exit of said composition of matter, C isan iron chelating moiety, and M is a functional-masking groupsusceptible to cleavage by (OH.).
 21. (canceled)
 22. A therapeuticcomposition comprising: (a) a compound of the formula R-L-C-M or apharmaceutically acceptable salt thereof; wherein R is H or abiocompatible moiety capable of facilitating or hindering thepenetration of said therapeutic composition in target cells, L is alinker connecting R to C, wherein L is capable of facilitating a rapidcellular intake and delaying a cellular exit of said therapeuticcomposition, C is an iron chelating moiety, and M is afunctional-masking group susceptible to cleavage by (OH.), and (b) apharmaceutical carrier.
 23. (canceled)